USPTO Patents Application 09842637

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INVESTOR IN PEOPLE

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Dated 9 May 2001

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9909962.4

' EA438S0-1 000027..
0 0.00 - 9?09%2.i

St. George's Enterprises Ltd

Cranmer Terrace

Tooting

London SW17 ORE

GB

Improvements in or relating to
screening antibacterial agents

Frank B. Dehn & Co

179 Queen Victoria Street
London
EC4V 4EL

166001

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I/We request the grant of a patent on the basis of this application.
Signature . Date 29 April 1999

John Christbpher Marsden
0171 7.06 0600

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68798/002.604

-Improvements in or relating to screening
antibacterial agents .

The present invention relates to the isolation of
subpopulations of stationary phase bacteria which
exhibit resistance to conventional antibacterial agents,
to the use of such resistant subpopulations in screening
processes . for the identification of new and improved
antibacterial agents, to novel antibacterial agents
identified thereby, and to therapeutic applications of
such antibacterials . .

Tuberculosis remains a serious disease throughout
the world and it has been estimated that deaths
resulting from tuberculosis account for 7% of the total
number of deaths from infectious diseases. As discussed
in US Patent No. 5,700/925, the vast majority of
individuals who become infected with MycoJbacteriiiin
tuberculosis do not develop symptomatic tuberculosis but
do exhibit a positive reaction to the tuberculin skin
test. In such infected hosts, some bacteria, persist in
a dormant or latent state in which they are
substantially resistant to antimicrobial drugs. Over a.
lifetime, up to 10% of these infected but asymptomatic
individuals may go on to develop tuberculosis, often
many years after primairy infection, when the dormant
bacilli become activated and start to grow. This can
result in pulmonary tuberculosis and other variant forms
of the disease. Factors which predispose towards
activation of the dormant organism and manifestation of
the diseased state include poverty, poor living
conditions, malnutrition, immune deficiency or immune
suppression.

In general in 'the treatment of tuberculosis,
antimicrobial therapy using antibacterial agents such as
rifampicin, isoniazid and/or pyrazinamide is relatively

- 2 -

successful , against .actively growing bacteria. Such
therapy is ineffective, however, against bacteria which
remain .dormant ^ or which, having, undergone a. growing
phase, re-enter a dormant phase . This .causes particular
5 problems in the clinical treatment of tuberculosis.

sufferers and carriers because the resistance of the
dormant organisms necessitates long term
chemotherapeutic care.. Such long term treatment,
typically of six months duration, is unsatisfactory
10 since it is expensive, may result in poor patient
compliance and may encourage the development, and
emergence of antibiotic resistant strains of bacteria
over and above the /^r. tuberculosis targetted during
therapy,

15 It is believed that most pathogenic bacteria for

example Staphyloccus a.ureus , Haemophilus influenzae,^
Streptpccus pyogenes Streptococcus gordonii and E,
coli, possess a similar substantially antibacterial
agent -resistant subpopulation which may act as a pool

20 ' for reinfection during or after chemotherapy. These
persistent: bacteria are usually drug-sensitive at
relapse, indicating that their resistance to
chemotherapy is phenotypic rather than genetic.

In order, to investigate whether the metabolism of

25 - such persistant bacteria is . switched off with no cell

division (i.e. is spore- like) or is active .(i.e. so that
the cells will contain markers of metabolism such as
mRNA) , we have studied AT. tuberculosis Ln.an in vitro
stationary phase model obtained by long term. culturing

30 of the. organisms in a microaerophilic gradient, in which
the .stationary, phase .organisms are viable. Such
stationary phase bacteria were found to be .resistant to
rifampicin at the normal minimum inhibitory. .
concentration (MIC) level, of 0.1 fig/ml.

35 The present invention is based on the. unexpected

and surprising finding that , whilst treatment .of such
stationary phase bacteria with, antibacterial agents . such

^ - 3 - .

as rifampicin at concentrations greatly exceeding the
minimum inhibitory concentration reduces plate counts to
zero colony forming units (CPU's), there remains a small
number of persistent organisms which are detectable by,
5 for example, broth dilution counting. These resistant
subpopulations are - phenotypically resistant to
rifampicin, since they become sensitive to rifampicin at
normal MIC levels upon resumption of growth.

Analogous studies using stationary phase JS. coli
10 and S. aureus model systems and treatment thereof with
kanamycin and ampicillin respectively have yielded
similar results.

Such phenotypically resistant subpopulations of
stationary phase bacteria, obtainable by treating
15 stationary phase bacteria with a high dosage of an
antibacterial agent, constitute one feature of the
present invention.

The nature of the antibacterial agent employed may
depend on the particular bacteria being investigated. "
20 The' use of antibacterial agents such as rifampicin which
target RNA polymerase," for example at concentrations of^
10^, 10^ or 10^ times the normal MIC level, has been
found convenient . In a representative embodiment of
■ this aspect of the invention, treatment of stationary
f- 25 phase'Af." tuberculosis at a level of 10^ bacteria/ml for
one day with 100 /xg/ml of rifampicin (MIC x 10^) resulted
in a phenotypically resistant subpopulation of 10^
bacteria/ml. In further embodiments, treatment of
stationary phase E\ coli or S. 'aureus (I. 10^ bacteria/ml)

3 0 with 50_ jLtg/mi Jcanamycin or 100 jLtg/ml ampicillin for' 3

days reisuTts in a phenotypically resistant subpopulation
of H 10^ bacteria/ml.

On the basis ^ that the phenotypically resistant
subpopulations of the invention mimic the behaviour of
35 dormant bacteria in vivo, they "constitute valuable tools
in screening procedures ' designed to identify potentially
valuable new antibacterial agents^ It will be

appreciated; that antibacterial agents having .'activity
against- such phenotypically resistant subpopulations may
have great therapeutic potential in the treatment .of
diseases such as tuberculosis in which dormant bacteria
provide a pool for reinfection.

Such processes / for example comprising the steps
of: ' . '

(i) growing a bacterial culture to stationary
phase ;

(ii) treating said stationary phase culture with
an antibacterial agent at a concentration and for a time
sufficient to kill growing bacteria, thereby selecting a
phenotypically resistant subpopulat ion; '

(iii) incubating a sample of said phenotypically
resistant subpopulation with test compounds; and

(iv) . assessing any antibacterial effects ' against
said phenotypically resistant subpopulation, constitute
a further feature of the invention.

Such a process facilitates the rapid screening, of
large numbers of compounds quickly and efficiently. The
compounds to be screened may be any chemical compounds
and may be -already known or may themselves.be novel.
Especially suitable candidate, compounds are the products
of combinatorial chemistry, phage display libraries,
.natural products, synthetic, semi -synthetic or natural
homologues, analogues, derivatives and precursors of
known antimicrobial agents or other pharmaceuticals.
Candidate compounds identified in such a. manner,, for
example exhibiting a minimum inhibitory concentration of
1 [ig/ml or less in respect of the resistant
subpopulation, may then be subjected to further
analysis, efficacy and toxicity tests etc.

In a further aspect, the present invention is
directed towards novel chemical compounds i which exhibit
bacteriostatic or bactericidal effects on an -
antibacterial agent .resistant subpopulation of
stationary phase bacteria,- e.g. a^ rif ampicin-resistant

subpopulation .of M: tuberculosis , a kanamycin-resistant
subpopulation of E. coli or an ampicillin-resistant
subpopulation of S. aureus. , "

In another aspect, the present invention is
directed towards a composition or formulation comprising
a novel antimicrobial agent identified by the process
described herein and a pharmaceutically acceptable
diluent or excipient.

In the clinical management of tuberculosis and
other disease states where the establishment and
existence of dormant bacteria is problematic, it may be
advantageous to administer a combination of
antibacterial agents which are respectively directed
towards the different growth phases of these organisms .
Thus, for example, an appropriate chemotherapeutic
approach to the treatment of tuberculosis would be to
administer to a patient one or more antibacterial agents-
directed against the growing or log-phase of the
microorganisms and one or more antibacterial agents
directed against the dormant or stationary phase
population.

Thus, a preferred formulation according to the
present invention comprises at least one antibacterial
agent which has activity against actively- growing
bacteria and at least one antibacterial agent .having
activity against the phenotypically resistant
subpopulation of the stationary phase of said bacteria .
Such formulations may be presented as a combined
preparation^ for simultaneous, separate or sequential use
in the treatment of bacterial infections such as
tuberculosis . -.^ - • -

In a further aspect, the present invention provides
the compounds identified' as effective against
phenotypically resistant subpopulations of stationary
phase bacteria by the above screening process for use in.
the treatment of bacterial infection.

^ In yet another- aspect, - the. present invention

relates to the use of compounds identified as effective
against phenotypically resistant subpopulations of
stationary phase bacteria by the above screening . process
in the preparation of a medicament for the treatment of
bacterial infection,

The use of such antibacterial compounds identified
by the above process in the treatment of bacterial
infections involving dormant bacteria constitutes
another aspect the present invention.

Alternatively viewed, the present invention
provides a method of treatment of a bacterial infection
.comp>rising administering to a patient in need of such
therapy an. effective, amount of an antibacterial agent
directed towards the stationary phase of growth/
optionally in the presence of one or more antibacterial
agents directed towards the growing phase of said
organism.

In each aspect of the invention, the stationary
phase bacteria which are the target of the the . ^
antibacterial agents may include . pathogenic bacteria
such as Staphylococcus aureus, Haemophilus influenzae,
Streptoccus. pyogenes. Streptococcus gordonil , Eschericia
coll and particularly preferably,- Mycobacterium ^
tuberculosis ,

The following non-limitative Examples serve to
illustrate the invention. In the accompanying drawings:

Fig. 1 shows growth curves for. E. coli and
aur*eus over a period of 10 days;

Figure 2 shows the viability of E. coli after
treatment with 50 ^xg/ml kanamycin during stationary
phase and log phase growth; .

Figure 3 shows the viability of S. aureus after
treatment with 100 fig/ml ampicillin. during stationary
phase and log phase growth.

Growth of M. tuberculosis and selecti on for a -
phenotvpicallv but not aeneticallY rif ampicin resistant
pQpul^tiou ot C^J-J-g

M. tuberculosis H3 7Rv was grown in Middlebrook. 7H9 broth
containing 0.05% Tween 80 supplemented with 10% ADC,
without agitation or other disturbance for up to '100
days, in accordance with the procedure of Wayne (1976)
Amer. Rev. Resp. Dis. 114: 807-811. Where the
stationary phase organisms were viable they were
resistant to MIC levels of 0.1 ;xg/ml rifampicin.'

Sixty 10 ml samples of the cultures were vortexed with
glass beads for 3-5 minutes, followed by sonication in a
water bath sonicator for less than 5 minutes . All the'
cultures were pooled into a 500 ml sterile bottle with
screw cap.

Prior to addition of rifampicin, samples of the culture
were taken to check purity, ' viability and metabolic
activity- as follows:

1. 2 X 100 ^1 for CFU counts
2.2 X- 5 ml for broth counts

3. 2 X 10 ml for [^H] uridine counts

4. 2x5 ml for [^^S] methionine counts

5. - 10 ml for RNA extraction for RT-PCR

6. 10 ml for drug- free control -

7. 20 fil for blood agar to check sterility

Selection and detection of a rif ampi cin-resistant
S Ub pQPU la t AQj Tt

50 ml of 1000 /xg/ml rifampicin was added to 500 ml of
the above culture to obtain a final rifampicin

concentration of 100 /xg/ml, and the culture was .\
incubated at 37°C for 5 days. The cells were then
harvested in sterile 5 0 ml tubes, by centrifugation at
SOOOg for 15 minutes, then washed twice in sterile PBS
containing 0.05% Tween 80 and Selectitab antibiotics
(which comprises a combination of antibacterial agents
which kills most bacteria but not M. tuberculosis , thus
preventing AT. tuberculosis from becoming overgrown by
faster growing bacteria) . The cells were resuspended in
500 ml fresh- 7H9 medium. .

Sterility of the culture was checked before - further
experiments. 2 x 100 /il samples of the culture was
added to blood agar plates in duplicate which were .
incubated at 3 7 °C overnight. The cell suspension was
kept at 4°C overnight. Contaminated cultures should.
always be discarded. ;

It was .found that this treatment with high levels of
rifampicin reduced plate counts to zero colony . forming
units but resulted in a small number of persisting
organisms which were detectable .by .broth . dilution
counting. Table 1 shows a typical e>cample of such
selection where 10^ bacteria/ml remained from 10^ after
one :day of ^ rifampicin treatment. , From this, it appears
that M. tuJbercuIosis has at least; two populations of
stationary phase organisms: the first is killed by high
dose rifampicin and the second 'persists* and is
phenotypically resistant. Upon resumption , of growth,
the rif ampicin-resistant stationary phase subpopulation
becomes sensitive to rifampicin at the normal MIC level
of 0.1 ' fig/ral . . .

- 9 -

EXAMPLE 2 ^ ^

Characterisatio n of- the rifa mpicin- resistant
gubpQp\iIqLtipn - —

5 . ■ ^

Samples of the rifampicin resistant subpopulation of
cells were analysed and the cell population further
characterised. The population was shown to be
metabolically active by virtue of transcriptional and
^-tIO translational activity detected in the population.

The cells were shown to be very responsive to changes in
their environment. When rifampicin was removed and
replaced by growth medium alone, the level of -

15 ' transcription of four genes analysed increased by 5- €o
10-fold after 12 hours. ' Radioactive uridine -
incorporation also increased about 5- fold after removal
of rifampicin and incubation with medium alone (Table 1
- compare counts at 5* and 5 days) . This suggested that

20 the- level of transcription increased rapidly under these
circumstances. The data do not exclude the possibility
that the organisms replicate in the presence of fresh"*'
medium, although this would only account for a^two- fold
rise in transcription in 12 hours, since the generation

25 time of M;- tuberculosis is -about 20 hours . The data
support a hypothesis that, in this in vitro model of
drug resistant statioriary-phase bacteria, there is a
major population of bacteria that are actively
transcribing RNA and are environmentally; reactive , yet

30 remain plate culture negative. . .

To distinguish between phenotypic and g-enotypic
resistance the bacteria thoroughly washed after
rifampicin treatment and cultured in liquid 7H9 medium
35 for 6 weeks. In four separate experiments M.

tuberculosis was invariably grown. These bacteria were
sensitive to 0.1 /xg/ml rifampicin and were negative for

. - 10 -

ri'fampicin resistant mutations in rpoB as detected by
RT-PCR and hybridisation with oligonucleotide probes
(Immunogenetics N.y . , Netherlands; data not^^
This indicates that the resistance is phenotypic in this
5 model.

It is unlikely that the mechanism of induced resistance
depends on mutation or reduced levels of expression of
rpoB mRNA (i.e. resulting in a reduced level of drug
10 target) because transcription continues even in the
presence. of rifampicin (Table 1) .

. _ Table 1 ...

15 . Incorporation of I^H] -uridine into AT. tuberculosis after
addition of rifampicin

Days in rifampicin Plate cputns Broth counts [^H] -uridine .

20

0

6.6 X 10^

10^

. 74682±630

1

0

10^

2228±88

2

0

10^

2316±120

3 .

. 102

2430+54

4

0

10=^

2318+126

0

102

2388±20

0 .

10^

518+10

0-

0 (heat-killed)

0

180±19

Legend , ^

30 ; . ,

M . tuJbejrculosis was grown in 7H9 medium, containing 0.05%
of Tween 80 supplemented with 10% ADS (Difco.
Laboratories) without shaking for. 100 days. - Rifampicin
was added to the cultures at a final- concentration, of

35 100. /ig/ml for 5 days.. . Viability was estimated at one
, . ._day intervals The cells were thoroughly washed and 100
/il of --samples from 10-fold dilutions of the cultures

- 11 -

were added to triplicate plates of Middlebrook 7H11
medium supplemented with OADC (Difco Laboratories) ,
Colony forming units (CPU's) were counted after -
incubation of. the plate for 3 weeks at 37°C. Broth
5 counts were performed at 10 -fold dilutions by adding 1
ml of the sample to 9 ml of 7H9 medium. Viability was
estimated by examining growth in the diluted cultures
after incubation for 6 weeks at 37°C.

For incorporation of radioactive uridine, 10 ml of

10 the culture for each time point was washed twice to

remove the remaining rifampicin and then resuspended in
10 ml 7H9 medium followed by incubation with 10 ^tCi/ml
of [^H] -uridine for 20 hours. For 5* the culture was not
washed after incubation with rifampicin and 10 /xCi/ml of

15 [^H] -uridine was added to the culture which was further*^

incubated in the presence of rifampicin for 20 hours.
The RNA was extracted by methods known in the art . The
RNA [^H] -uridine incorporation was determined as counts
per minute of trichloroacetic acid precipitated RNA.

20 , The results were confirmed in three independent
experiments.

Susceptibility assessment of drug libraries

25 Sterile 0 . 7 ml labelled transparent plastic snap-capped
tubes containing 1-20 /xg of the compound to be tested
are used. The compound is dissolved in 0.25 ml of
sterile distilled water or other appropriate diluent.
0.25 ml of the rifampicin treated culture is added to

3 0 each tube in the class I cabinet in a category III

safety containment laboratory with care - being --taken to
avoid contamination.^ The tubes are ^incubated at 37°C.
The drug-effects are examined by CFU counts by addition
of 2 X 50 ptl" of each' sample to 7H11 agar plates in

35 duplicate including 2 drug-free controls at 1 week

' interval's . ' A series of 10-fold dilutions of ^ the samples
may be required, which is made in 7H9 broth with 0 '. 05%

- 12 -

Tween 80 but vwithout ADC, then the diluted samples are
plated on 7H11 agars and the concentration effect of the
candidate compounds determined... ...

EXAMPLE 3 .

Selection of ph enQtyicallv but not aenotypically
resistant subpo pulations of E. coli and S, aureus using
kanamycin and ampicillin respectively

Growth of Escherichia coli and Staphylococcus aureus

Escherichia coli K12 and Staphylococcus, aureus are grown
in 10 ml of nutrient, broth No. 2 (Oxoid) with continuous
shaking at 120 rpm for 10 days. Viability of the
bacteria is estimated by colony forming unit counts at 2
hours intervals for the first 24 hours and 12-24 hours
afterward. From serial 10 -fold dilutions of the
experimental cultures , . 100 ^xl samples are added to
triplicate plates of nutrient agar plates (Oxoid) .
Colony forming units (CPU) are counted a:fter incubation
of the plates at 37°C for 24 hours.

Selection of pe rsistent bacteria bv antibiotics

Ampicillin and kanamycin are added to 5 -day stationary-
phase cultures of E, coli and S, aureus respectively to
a final concentration of 100 /xg/ml and 50 fig/ml
^respectively for 3 days. After 3 days of antibiotic
treatment, the cells are washed with sterile distilled
water 3 times, then resuspended in 10 ml fresh nutrient
broth. Viability is estimated by CPU counts and broth
dilution counts. Broth dilution counts are performed in
a serial 10-fold dilution in nutrient broth, then 1 ml
of each dilution in added into 9 ml of nutrient broth in
30-ml universal tubes in triplicate. The growth curves
for E. coli and S. aureus over the 240 hour period in

- 13 -

the absence of antibiotics is shown in Fig. iv The
effect of antibiotic exposure on the E. coli and S.
aureus during both log and stationary phase of growth is
shown in Figs, 2 and 3. Treatment of the cultures
5 "during stationary phase results in the selection of an
antibiotic resistant subpopulation of cells.

- 14 -

Claim s - - > ' - - '

1. A phenotypically antibiotic-resistant subpopulation
of stationary phase bacteria, obtainable by treating
stationary phase bacteria with a high dosage of an

ant ibacberi^^ .^g?Ii?^_.„__ _ ^ _„_.^. _^„....

2. A phenotypically antibiotic-resistant subpopulation
of stationary phase bacteria as claimed in claim 1
wherein said bacteria are Staphylococcus aureus,
Eschericia coli, Haemophilus influenzae, Streptoccus
pyogenes , Streptococcus gordonii or. Mycobacterium
tuberculosis.

3. A process for screening for agents having
antibacterial activity against stationary phase bacteria
comprising the steps of:

(i) growing a bacterial culture- to stationary
phase;

(ii) treating said stationary phase culture with
an antibacterial agent at a concentration and for a time
sufficient' to kill growing bacteria, thereby selecting a
phenotypically resistant subpopulation;

(iii) incubating a sample of said phenotypically
resistant subpopulation with test compounds; and

(iv) assessing any antibacterial effects against
said phenotypically resistant subpopulation,

4. A process as claimed in claim 3 wherein said
bacteria are MycoJbacteriuzn tuberculosis and said
antibacterial agent is rifampicin.

5. A process as claimed in claim 3 -wherein said
bacteria are Eschericia coli and said antibacterial
agent is kanamycin.

6. A process as claimed in claim 3 wherein' said '

- 15 -

bacteria are Staphylococcus aureus and said
antibacterial agent is ampicllin.

7. Chemical compounds which exhibit antibacterial
5 activity against a phenotypically antibiotic resistant

subpopulation of bacteria as defined in claim 1 or claim

2".

a. A composition comprising an antibacterial agent as
10 defined in claim 7 and a pharmaceutically acceptable
excipient or diluent.

9. A formulation comprising at least one. antibacterial
agent having activity against actively growing bacteria

15 and at least one antibacterial agent having activity /;
against a phenotypically antibiotic-resistant ^
subpopulation of stationary phase bacteria wherein /said
formulation is presented as a combined preparation- for
simultaneous, separate or sequential use in the

20 treatment of bacterial infections. :

10. ^ The use of antibacterial compounds . as defined in v
claim 7 in the treatment of bacterial .infections ,
involving dormant bacteria. \ ■

11 . Use of antibacterial compounds as, defined in claim
7 in the preparation of a medicament for the treatment
of bacterial infections involving dormant bacteria.

3 0 12. :A method of treating of a bacterial infection ■

comprising administering to- a patient in- need of . such
therapy an effective amount of an antibacterial compound
as def ined in claim . 7.

35

13. A method as claimed in claim 12. further comprising
administration of one or more antibacterial agents
directed ' towards growing bacteria . ^ ;

1/3

6.S

6

20 40

^ io 100 120 140 160 180 200 220 240

a r s of Incubation

Fig. 1

stationary phase Log P^ase

Fig. 2

i

3/3

Viability Of Staphylococcus aureus after Treatment with Antibiotics

Fig. 3

Anthony R.M. COATES
' USSN 09/842, 63.7
Filed: April 27, 2001

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